1 Development of Immunoassays for the Detection of Markers of Human and Environmental Exposure Department of Entomology University of California @ Davis.

1

Development of Immunoassays for the Detection of Markers of Human and Environmental Exposure

Department of EntomologyUniversity of California @ Davis

[email protected]

Bruce D. Hammock, Ph.D.

Shirley J. Gee

2

UC Davis/NIEHS Superfund Hazardous Substances Basic Research Program

“Biomarkers of Exposure to Hazardous

Substances”

University of California, Davis

3

1. Transport, Transformation and Remediation of Perchlorate and VOCs in the Vadose Zone

2. Aquatic Biomarkers in Site Characterization and Remediation

3. Development and Implementation of Immunoassays for Human and Environmental Monitoring

4. Biomarkers of Exposure to Pulmonary Toxicants

5. Development and Applications of Integrated Cell-Based Bioassays

6. Assessing Adverse Effects of Environmental Hazards on Reproductive Health in Human Populations

7. Thermal Remediation

8. Development of Rapid, Miniaturized Sensors for Use in the Detection of Environmental Toxins

9. Epidemiology Studies

PROJECTS

4

A. Analytical Chemistry Core

B. Statistical Analysis of Toxics Measurements

C. DNA Microarray Core

D. Training Core

F. Administrative Core

CORES

5

RESEARCH TOPICS ADDRESSED BY PROJECTS

• ANALYTICAL METH 1, 3, 5, 8, A, B

• BIOMEDICAL 2, 3, 4, 5, 6, 9

• BIOSTATISTICS B, C

• BIOINFORMATICS 1, 2, 3, 4, 5, B, C

• CBPI 6, 9, E

• ECOLOGY 1, 2

• ENGINEERING 1, 7, 8

• EPIDEMIOLOGY 6, 9

• HYDROGEOLOGY 1

• MECHANISM 1, 2, 4, 5, 6

• MICROBIOLOGY 1

• METABOLISM 1, 2, 3, 4, 5, A

• MOLECULAR BIOL 1, 2, 4, 5,

• REMEDIATION 1, 7

• ON SITE PROJECTS 1, 9

• TOXICOLOGY 3, 4, 5, 6, A

• TRANSPORT 1, 2

6

O

O

O

Cl

ClCl

Cl Cl

ClCl

Cl

Cl

Cl Cl

Cl

Cl

2,3,7,8-Tetrachlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzofuran 3,4,3',4,'5-Pentachlorobiphenyl

Halogenated Aromatic Hydrocarbons

7

Chemical and Biological Techniques to Detect and QuantitateHalogenated Dioxins and Related Chemicals

Instrumental Immunoassay Bioassays

Biological/Toxic Potency Estimates of a Complex Mixture are Based on TCDD Equivalency Factors (TEFs) for the Specific

Chemicals Present in the Mixture.

TEFs are Derived from the Relative Toxic Potency of each HAHand is Directly is Related to their Relative Ability to Activate

the Ah Receptor (AhR) and AhR Signaling Pathway

8

ELISAProject 3

GLC-MSCore A

CALUXProject 5

TCDDor

UNKNOWN

ELISAProject 3

GLC-MSCore A

BIOASSAY DRIVENFRACTIONATION

Core A

ARBProject

1

USGSProject

2

SERUMProject

6

SOOTProject

7

9

ANALYTICAL PROBLEMS

• ANALYTICAL COSTS TOO HIGH

• ANALYSIS TIME TOO LONG

• ANALYSIS NOT FIELD PORTABLE

• ANALYSIS NOT IN ANALYSTS’ HANDS

• NOT ADAPTABLE TO MULTIPLE ANALYTES

10

TYPICAL ANALYTICAL COSTS

ATRAZINE

PARAQUAT

GLYPHOSATE

TCDD

$50 - $150/Sample

$200 - $500/Sample

$500 - $1000/Sample

$1,500 - $5,000/Sample

11

ADVANTAGES

• Sensitive

• Selective

• Rapid

• Cost Effective

• Applicable

• Adaptable

12

DISADVANTAGES

• Sensitivity

• Cross reactivity/Interferences

• Reagent availability

• New technology

• Large sample load required

• Difficult to apply to multianalyte analysis

13

A B C D1

2

3

4

Absorbance

Log Concentration

Polystyrene

Coating Antigen

E Enzyme coupled to Second Antibody

First antibody

Analyte

Substrate to Product

A

B

CD

14

Immunoassays Developed

• Triazines– Atrazine– Simazine– Hydroxytriazines– N-Dealkylated triazines– Atrazine mercapturate

• Thiocarbamates– Molinate– Thiobencarb

• Paraquat• Amitrole• Bentazon• Bromacil• Carbaryl• Diflubenzuron

– Other benzoylphenyl ureas• Fenoxycarb• Glyphosate• Alternaria toxins• Bacillus thuringiensis -endotoxin• Bacillus thuringiensis -exotoxin• 2,4,5-Trichlorophenoxyacetic acid

• Trichlopyr• Triton Series X and N detergents• Octyl and nonyl phenol• Urea Herbicides

– Monuron– Diuron– Linuron

• Naphthalene and metabolites• Nitrophenols (other nitroaromatics)• Pyrethroids

– Fenpropathrin– Esfenvalerate– Permethrin– Deltamethrin– 3-Phenoxybenzoic acid– Metabolites and conjugates

• Dioxins• General Mercapturates

– S-benzyl mercapturate• General Glucuronides

– Phenyl glucuronide– Thiophenyl glucuronide

15

Cl

ClO

O

CN

O

O

O

CN

O

ClO

O

CN

O

F

Cl

Cl

O

OO

Cl

Cl

Major Pyrethroids Used in California

Permethrin (57.5%) Cypermethrin (22.6%)

Cyfluthrin (7%) Esfenvalerate (6%)

*In 1998, a total of 650,000 lbs pyrethroids were applied in California

16

O O

O Cl

Cl

OO

O Cl

Cl

H2N

Hapten Design for Compound-specific Assay (I)

Target Compound Hapten

Permethrin

• Close mimic of analyte• Distal attachment site• Four carbon handle optimal

17

Hapten Design for Class-specific Assay

Target Compound Hapten

OO

OCOOH

OO

O

COOH

CN

O

O

HO

O

CHO

Type I Pyrethroids(without CN)

Type II Pyrethroids(with CN)

All Pyrethroids(with PB)

18

% C

ontr

ol A

bsor

banc

e

0.001 0.01 0.1 1 10 100 1000 100000

20

40

60

80

100

Concentration (ppb)

permethrinI50 = 5 ppbLDL = 0.001 ppb

Standard Curve for Permethrin

19

020406080

100120

Perm

ethrin

Phenoth

rin

Cyper

met

hrin

Esfen

vale

rate

Delta

met

hrin

Cyflu

thrin

Fenpro

pathrin

PBA

Cro

ss-r

eact

ivit

y %

Permethrin-specific Immunoassay (Selectivity)

OO

OC l

C l

Permethtrin

I50 = 2.5 ppbLOQ = 0.01 ppb (SPE)

ni ni ni ni

*ni = less than 10 % inhibition at 10 ppm

20

Relationship between Permethrin Measured by ELISA and GC-MS

Y = 1.211x - 0.068 R2 = 0.900, n = 27

Permethrin (GC-MS, ppb)

0.001

0.01

0.1

1

10

100

0.001 0.01 0.1 1 10 100

y = x

Per

met

hri

n (E

LIS

A, p

pb)

21

Role of Immunochemistry in Trace Analysis

• Direct immunochemical analysis• Prioritization of samples for other methods• Immunoaffinity cleanup for other methods• Post separation detection• HPLC/Microbore LC• Capillary electrophoresis• TLC

22

ELISA: A Complementary Technology

ELISA: A Complementary Technology

Analytical Chemist

MS GC CE HPLC

IR SFC UV/VISImmunoassay

23TCDD (pg/well)

0.001 0.01 0.1 1 10 100 1000 10000

Ab

so

rban

ce

0.0

0.1

0.2

0.3

0.4

0.5

0.6

O

O

Cl

Cl

Cl

Cl

IC50 = 4 pg/well

24

Strategies for detection in Strategies for detection in environmental samplesenvironmental samples

• Increased throughput• Increased sensitivity• Increased specificity

• Small volumes• Minimized pre-processing of samples• Minimized autofluorescence

Achieved by

Emission spectra of lanthanides

25

Department of Environmental ToxicologyUniversity of California @ Davis

[email protected]

Development, Validation and Application of Recombinant CellBioassay Systems for Rapid Detection of Dioxins and

Related Halogenated Aromatic Hydrocarbons

Michael S. Denison, Ph.D.

26

Chemical and Biological Techniques to Detect and QuantitateHalogenated Dioxins and Related Chemicals

Instrumental Immunoassay Bioassays

Biological/Toxic Potency Estimates of a Complex Mixture are Based on TCDD Equivalency Factors (TEFs) for the Specific

Chemicals Present in the Mixture.

TEFs are Derived from the Relative Toxic Potency of each HAHand is Directly is Related to their Relative Ability to Activate

the Ah Receptor (AhR) and AhR Signaling Pathway

27

Spectrum of Toxic and Biological Effects Producedby TCDD in Different Species and Tissues______________________________________________

• Immunotoxicity• Hepatotoxicity• Wasting Syndrome• Tumor Promotion• Dermal Toxicity (Chloracne)• Teratogenicity• Lethality• Uroporphyrin Accumulation (Porphyria)• Endocrine Disruption• Modulation of Cell Growth, Proliferation and

Differentiation• Induction of Gene Expression

Cytochrome P4501A1/2 and 1B1UDP-Glucuronosyl Transferase 1*06Glutathione S-Transferase YaQuinone Reductase-Aminolevulinic Acid SynthaseProstaglandin Endoperoxide H Synthase 2

The biological and toxicological effects of TCDD and related halogenated aromatic hydrocarbons (HAHs) are mediated by the Ah Receptor (AhR)

28

AhR Signal Transduction Pathway

TranslationNew Polypeptides

Increased Cytochrome P-4501A1

ARNT

Exogenous and Endogenous Ligands

Endogenous Ligands

Other Gene Products

mRNA

DREs CYP1A1

DREs Other Genes

Other Factors?Translocation

Proteosome

Degradation

AhR hsp90

XAP2 hsp90p23

Toxicity

29

AhR Signal Transduction Pathway

TranslationNew Polypeptides

Increased Cytochrome P-4501A1

ARNT

Exogenous and Endogenous Ligands

Endogenous Ligands

Other Gene Products

mRNA

DREs CYP1A1

DREs Other Genes

Other Factors?Translocation

Proteosome

Degradation

AhR hsp90

XAP2 hsp90p23

Toxicity

30

Development of a Ah Receptor-Based Bioassay Bioassay Systemfor Detection and Relative Quantitation of Dioxin and Related HAHs

TCDD + AhR

TCDD:AhR

TCDD:AhR*

TCDD:AhR:DRE

TranscriptionalActivation

Ligand Binding

Transformation

DNA Binding

Gene Expression*

31

CALUX (Chemically-Activated Luciferase Expression) Cell Bioassay

TranslationNew Polypeptides

Increased Cytochrome P-4501A1

ARNT

TCDD HAHs PAHs

Luciferase Activity

mRNA

DREs CYP1A1

DREs Luciferase

Other Factors?Translocation

AhR hsp90

XAP2 hsp90

32

CALUX Bioassay Procedure

H1L6.1c3 Mouse HepatomaCells Plated into 96-Well

Microplates

Chemicals Added to Each Well and

Incubated for 24 hours

Wells are Washed, CellsLysed, and Luciferase

Activity Measured in a Microplate Luminometer

33

10 -710 -810 -910 -1010 -1110 -1210 -1310 -140

1

2

3

4

5

6

7

TCDD Concentration (M)

Lu

cif

era

se A

cti

vit

y (

RLU

/ug

pro

tein

)

TCDD Dose Dependent Induction of Luciferase Activityin Stably Transfected Mouse Hepatoma (Hepa1c1c7) Cells

EC50 = 10 pMMDL = 1 pM

Current Microplate AssayMDL = 0.03pg/assay

34

10 -510 -610 -710 -810 -910 -1010 -1110 -1210 -1310 -140

2

4

6

8

2378-TCDD

2378-TCDF

23478-PCDF

12478-PCDD

3344-TCB

33445-PCB

Chemical Concentration (M)

Lu

cif

era

se A

cti

vit

y

(R

LU

/ug

pro

tein

)

Activation of the CALUX Cell Bioassay by PCDDs, PCDFs and PCBs

35

WHO Toxic Equivalency Factors (TEFs) and CALUXRelative Potency (REP) Factors for Chlorinated Dibenzo-

p-Dioxins, Dibenzofurans and Biphenyls._______________________________________________

Compound WHO-TEF CALUX REP

_______________________________________________

2378-TCDD 1 1.00 ± 0.0112378-PeCDD 1 0.73 ± 0.1123478-HxCDD 0.1 0.075 ± 0.014123678-HxCDD 0.1 0.098 ± 0.017123789-HxCDD 0.1 0.061 ± 0.0121234678-HpCDD 0.01 0.031 ± 0.008OCDD 0.0001 0.00034 ±0.00008

2378-TCDF 0.1 0.67 ± 0.0112378-PeCDF 0.05 0.14 ± 0.0423478-PeCDF 0.5 0.58 ± 0.08123478-HxCDF 0.1 0.13 ± 0.02123678-HxCDF 0.1 0.14 ± 0.03123789-HxCDF 0.1 0.11 ± 0.02234678-HxCDF 0.1 0.31 ± 0.061234678-HpCDF 0.01 0.024 ± 0.0071234789-HpCDF 0.01 0.044 ± 0.010OCDF 0.0001 0.0016 ± 0.0005

PCB 77 0.0005 0.0014 ± 0.0004PCB 81 0.0001 0.0045 ± 0.0012PCB 114 0.0005 0.00014 ± 0.00002PCB 126 0.1 0.038 ± 0.007PCB 156 0.0005 0.00014 ± 0.00002PCB169 0.01 0.0011 ± 0.0003_______________________________________________

36

Bioassay Systems - Considerations

Structural Diversity of AhR Agonists and/or Antagonists:Potential for False Positives in HAH Detection

O

O

O

O

O

Cl

ClCl

Cl Cl

ClCl

Cl

Cl

Cl Cl

Cl

Cl

2,3,7,8-Tetrachlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzofuran 3,4,3',4,'5-Pentachlorobiphenyl

CH3

3-Methylcholanthrene -Naphthoflavone Benzo(a)pyrene

Halogenated Aromatic Hydrocarbons and Polycyclic Aromatic Hydrocarbons

O

O

Cl

Cl

N

N

N

H

2

O

H

Guanabenz

N

H

2

S

C

H

3

2(Methylmercapto)aniline

N

H

2

N

H

2

1,5-Diaminonaphthalene

N

N

S

N

O

CH2

OCH3

CH3CH3

Omeprazole

CH3O

N

C

H

3

H

5-Methyl-2-phenylindole

N

H

2

O

H

(1S,2R)-(-)-cis-1-Amino-2-indanol

N

H

N

H

N

H

2

N

O

2

O

2

N

O

C

H

3

SRN-P2:109,NH2

N

N

H

2

C

H

3

1-Methyl-1-phenylhydrazine

N

N

N

H

CN

CF3

SKF71739

S

N

C

H

3

N

H

2

2-(4-Amino-3-methylphenyl) benzothiazole

N

S

S

O

N

O

O

S

Cl

ClCl

Cl

2,3,7,8-Tetrachlorodibenzo-p-dioxin

CH3YH439

CH3

CH3

H

• Promiscuous ligand binding by the AhR requires appropriate extraction and chemical clean-up methods to remove unwanted compopunds

37

Flow Diagram for Analysis of Samples by CALUX and GC/MS

Sample

Extraction and Chemical Clean-up

CALUX Bioassay Analysis

Positive

Negative

No Compounds that Activate the Ah Receptor and/or

Contains Compounds that Block Activation (i.e. Antagonists)

Analysis by HRGC/MS

Estimate of relative

activity

Total HAHsPCDD/PCDF

&PCB

38

Bioassay Systems - Considerations • Calculation of relative biological potency of an unknown sample extract

10 -710 -810 -910 -1010 -1110 -1210 -1310 -140

1

2

3

4

5

6

7

TCDD Concentration (M)

Resp

on

se

10 110 210 310 410 5106

10 710 80

1

2

3

4

5

6

7

Dilution Factor

EC50 = 20pM

ED50 = 1000-fold dilution = 20pM Thus, the original sample contains the equivalent (TEQ) of 20nM TCDD

EC50

EC50 = 1000-fold dilution = 20pMThus the original extract contains the

equivalent (bioassay-TEQ) of 20 nM TCDD

39.

Applications of the CALUX Dioxin Cell Bioassay

1. Biological Samples• Blood (whole serum and extracts)• Breast Milk• Tissue Extracts

2. Environmental Samples• Soil and Sediment• Ash• Emission (PUF)• Pulp and Paper

3. Food Samples• Animal Fats (oil and fats)• Milk and Butter• Animal Feeds

40

Comparative GCMS/CALUX Results fromthe Umea Round Robin Analysis of

Environmental Samples for ChlorinatedDibenzo-p-Dioxins, Dibenzofurans and

Biphenyls.

______________________________________ TEQ (ng/g sample)

Sample GC/MS CALUX______________________________________

Soil A 0.3 0.16

Soil B 0.17 1.2Soil C 0.19 0.6

Soil D 0.30 1.06

Ash A 0.40 0.13

Ash B 0.04 0.04Ash C 0.42 0.50

Solution F 222 448Solution H 3.42 8.39

______________________________________

41

Double-Blind Validation ResultsComparison of CALUX and HR GC/MS Analysis of Ash and Exhaust Gas Samples

R2 = 0.947

0.01

0.1

1

10

100

1000

10000

100000

1 10 100 1000 10000 100000

CALUX, pg TEQ/gram

R2 = 0.918

0.0001

0.001

0.01

0.1

1

10

100

1000

0.01 0.1 1 10 100 1000

CALUX, pg TEQ/m3

Ash

Exhaust Gas

42

Double-Blind Validation Results Comparison of CALUX and HR GC/MSAnalysis of Soil and Human Fat Samples

R2 = 0.854

0.01

0.1

1

10

100

1000

0.1 1 10 100 1000

CALUX, pg TEQ/gram

HR

GC

MS

, pg

TE

Q/g

ram

R = 0. 8051

0

20

40

60

80

100

0 20 40 60 80 100CALUX Assay(pgTEQ/ g fat)

HRGC

MS(p

gTEQ

/g f

at)

Soil

Human Fat

43

Environmental Samples Contain Additional Ah Receptor Active HAHs?

R2 = 0.947

0.01

0.1

1

10

100

1000

10000

100000

1 10 100 1000 10000 100000

CALUX, pg TEQ/gram

R2 = 0.918

0.0001

0.001

0.01

0.1

1

10

100

1000

0.01 0.1 1 10 100 1000

CALUX, pg TEQ/m3

Ash

Exhaust Gas

R2 = 0.854

0.01

0.1

1

10

100

1000

0.1 1 10 100 1000

CALUX, pg TEQ/gram

HR

GC

MS

, pg

TE

Q/g

ram

Soil

R = 0. 8051

0

20

40

60

80

100

0 20 40 60 80 100CALUX Assay(pgTEQ/ g fat)

HRGC

MS(p

gTEQ

/g f

at) Human Fat

44

Bioassay Systems - Further Considerations • Calculation of relative biological potency of an unknown sample extract• Promiscuous ligand binding by the AhR requires appropriate extraction and chemical clean-up methods for each bioassay

• Discrepancy between bioassay and instrumental analysis needs to be resolved a. Use of bioassay-specific REPs for TEQ estimates from instrumental analysis b. False positives or other AhR-active HAHs

R2 = 0.918

0.0001

0.001

0.01

0.1

1

10

100

1000

0.01 0.1 1 10 100 1000

CALUX, pg TEQ/m3

45

101.1.01.001.0001.0001

.001

.01

.1

1

10

WHO TEF Values for Dioxin-Like HAHs

CA

LU

X R

EP

Valu

es f

or

Dio

xin

-Lik

e H

AH

s

Comparison of GC/MS TEF and CALUX REP Values

46

Tiered Flow Diagram for Sample Screening

POSITIVERun immunoassay

NEGATIVENo compounds that bind

productively to Ah receptor

POSITIVERun GC/MS for

confirmation

NEGATIVENo TCDD-like

compounds

BOTH NEGATIVEUse CALUX to drivechemical purification

PCBELISA

TCDDELISA

CALUX

POSITIVERun GC/MS for

confirmation

NEGATIVENo TCDD-like

compounds

ELISA Also suggeststhat there are more 2,3,7-PCDD/Fs thanGC/MS indicates.

What could it be?

47

O

O

O

O

Br

O

H

Br

Br

Br

Br

Halogenated Aromatic Hydrocarbons

Cl

ClCl

Cl Cl

ClCl

Cl

2,3,7,8-Tetrachlorodibenzo-p-dioxin 2,3,7,8-Tetrachlorodibenzofuran Polybrominated Diphenyl Ether

O

Br

Br

Br

Br

Br

O

Br

Br

Br

Br

Br

O

Polybrominated Dibenzofurans (PBDFs)Polybrominated Dibenzo-p-dioxins (PBDDs)

Sources:Formed during PDBE SynthesisBurning of PBDE Containing MaterialsPhotochemical Reactions of PBDEs

One Possibility for the Higher Equivalent Estimates: PB/CDDs/Fs

2,3,7,8-PBDD/F found in PBDE workers@ n.d - 500pg/g blood lipid.

Mo-OctBDF produced from combustion of DBDE-polypropylene matrices

48

0

2000

4000

6000

8000

10000

12000

1,E-13 1,E-12 1,E-11 1,E-10 1,E-09 1,E-08 1,E-07 1,E-06 1,E-05

Concentration, M

RL

U2378-TCDD, n=11PCB 169, n=6PBB 169, n=52378-TBDD, n=4

Brominated Dioxins and Related HAHs are Relatively Potent Activators

49

In Vivo ED50 Values for Toxic and Induction Effects of Select PHDDs in the Rat

Congener In Vivo ED50 (µmol/kg body weight)Body Weight Loss Thymic Atrophy Gene Induction

2,3,7,8-TeCDD 0.05 0.09 0.0042,3,7,8-TeBDD 0.068 0.034 0.0076

1,2,3,7,8-PeCDD 0.62 0.17 0.0311,2,3,7,8-PeBDD 0.87 0.39 0.025

2,3-DiB-7,8-DiCDD 0.012 0.0073* 0.00049*2-B-3,7,8-TriCDD 0.12 0.035 0.0025

1,2,4,7,8-PeCDD 34.0 11.2 2.821,2,4,7,8-PeBDD 12.9 6.17 0.195Immature male Wistar rats (n=4), 14 days after a single intraperitoneal dose.Adapted from IPCS Env. Health Criteria #205 - data from Mason et al. (1987); Safe et al. (1989).

50

AhR Gene Expression Cell Bioassay Systems

Advantages

• Sensitive, relatively rapid and easy to carry out• Amenable to high throughput analysis• Relatively inexpensive compared to instrumental analysis• A significant amount of validation data is available for the CALUX bioassay • Overestimate TCDD equivalents in environmental samples (new dioxin-like HAHs?)

Disadvantages/Limitations

• Experience in cell culture techniques necessary• Proper sample cleanup methods needed • Instrumentation - Luminescent microplate readers, cell culture facilities• Overestimate TCDD equivalents in environmental samples (false positives?)• Extracts containing chemicals that are directly toxic to cells can not be analyzed• Synergistic/antagonistic effects (over/underestimate potency)

51

Bioassay Systems - Further Considerations • Calculation of relative biological potency of an unknown sample extract

• Promiscuous ligand binding by the AhR requires appropriate extraction and chemical clean-up methods for each bioassay (in vitro assays require more extensive clean-up methodologies)

• Discrepancy between bioassay and instrumental analysis needs to be resolved a. false positives or other AhR-active HAHs b. Use of bioassay-specific REPs for TEQ estimates from instrumental analysis

• Toxic Equivalency Factors (TEFs) versus Relative Potency (REPs) Values TEQs (instrumental) versus Bioassay-TEQs

• Need method(s) to correct for extraction and clean-up efficiency and recovery since samples can’t simply be spike with AhR-active HAHs like 2,3,7,8-TCDD

• Establish quality control criteria for bioassay methodologies

• Require full validation studies (versus instrumental analysis) for different matrices

52

1.1.01.0010

2000

4000

6000

8000

10000

12000

Day 1Day 14

TCDD (ng/ml)

Luci

fera

se A

ctiv

ity (

RLU

/well)

Microplates containing cells can be sealed and stored at room temperature for up to 14 days with little loss of TCDD-inducible luciferase activity.

Viewplate microplate with well sealer

Seal

Cell Bioassay Systems - Development

This development now allows plates to be prepared off-site and mailed to the site for extract treatment and analysis.

53

Application and Utilization of Bioanalytical Methods for Dioxin Analysis

• Detection, quantitation and chemical identification of dioxin-like chemicals in a variety of matrices including:

• Environmental samples (soil, water, air)• Biological (blood, milk, tissues)• Food and feed• Commercial and consumer products

• Determination of the effectiveness of bioremediation, biodegradation and contamination clean-up procedures for dioxin-like chemicals.

• Identification and characterization of other classes of dioxin-like chemicals.

54

Acknowledgements and Support

University of California @ DavisMichael Ziccardi, Joe Rogers, Patricia Garrison,

Bruce Hammock, Shirley Gee, Guomin Shan

Xenobiotic Detection SystemsGeorge Clark, David Brown, Mick Chu

Hiyoshi Corporation (Japan)Hiroshi Murata

Scientific Institute of Public Health, Brussels, BelgiumLeo Goeyens, Ilse van Overmeire

National Institutes of Environmental Health Sciences (NIEHS) Superfund Basic Research Program - ESO4699, ES04911

55

Ian Kennedy

Department of Mechanical and Aeronautical Engineeringand

Biomedical and Electrical Computer Engineering Graduate Groups

University of California Davis

Supported by the NIEHS Superfund Basic Research Program andNSF NNI

Ian Kennedy

Department of Mechanical and Aeronautical Engineeringand

Biomedical and Electrical Computer Engineering Graduate Groups

University of California Davis

Supported by the NIEHS Superfund Basic Research Program andNSF NNI

Nanotechnology and Biosensors

Nanotechnology and Biosensors

56

Strategies for detection in environmental samplesIncreased throughputIncreased sensitivityIncreased specificity

Achieved by Small volumesMinimized pre-processing of samplesMinimized autofluorescenceNew fluorescent labels

57

Competitive immunoassays in microdroplets using fluorescence quenching

Microdroplet cavity resonances

Application of the microdroplet approach to analysis on a chip

Development of new labels using novel materialsQuantum dotsEncapsulated lanthanide oxidesAbsorbing labels such as C60

Novel technologies

58

Quenching fluoroimmunoassay in microdroplets

Antibody quenching

Competitive Quenching Fluoro ImmunoAssay (QFIA)

- TCP

- TCP- F

analyte

Labeled analyte

59

Assay for TCPAssay for TCPCalibration curve for 2,4,6-TCP in PBS and in urine 1/50; 10 nM Af; 2.5 mg/ml Ab43; incubation time 45 min at room temperature

TCP, M

(I / I

AF)

*100

10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -440

50

60

70

80

90

100

110PBSurine 1/50

g/L urine

IC50 22.5

LOD 2

g/L ELISA QFIA

200 L

QFIA

microdrop

IC50 2.74 4.2 0.45

LOD 0.2 0.36 0.04

60

Intense optical field created by internally reflected fluorescence from Rhodamine

Micro cavity resonances

61

Intensities within droplets greatly reduced when C60 is

added to droplet

Images of Rh emission quenched by C60

62

Single E. coli detected using optical resonances without labeling

Couple to immunoassay for specificity or use mode structure for information

Detecting bacteria

63

Microdroplets on a chip

Biosensor chip

MIcrochannel filled with lowrefractive index silicon oil

Microdroplets ofaqueous sample

64

Trapped microdroplet with long interrogation time

65

Sharp emission spectrum increases spectral sensitivity

Long lifetime emission permits gated detection

Surface treatment prevents quenching

No need for chelation

Magnetic moments useful for separation and for imaging contrast agents

No photobleaching

Lanthanide oxide nanoparticles

66

Lanthanide oxides as reporters for bioassays

57

La

58

Ce

59

Pr

60

Nd

61

P

m

62

Sm

63

Eu

64

Gd

65

Tb

66

Dy

67

Ho

68

Er

69

T

m

70

Yb

71

Lu

67

Europium oxide spectra

80x103

70

60

50

40

30

20

10

Inte

nsi

ty (

arb

itra

ry u

nit

s)

650600550500450400350

Wavelength (nm)

Excitation spectrumobserved at 610 nm Emission spectrum

excited at 466 nm

4x106

3

2

1

Inte

nsi

ty (

arb

itra

ry u

nit

s)

600580560540520500

Wavelength (nm)

Excitation spectrum observed at 548 nm

Emission spectrum excited at 524 nm

Europium

Rhodamine

68

Gas phase synthesis and functionalization

AntibodyAntigen

A functionalized nanoparticle containing lanthanide oxide can be used in an immunoassay that takes advantage of the specific binding between an antigen and a homologous antibody.

69

Pure Eu2O3 particles

Photoluminescence Spectrum: Dominant Peaks at 615 and 623 nm; Short Lifetime Due to Concentration

Quenching Combined with Small Size.

Pure Eu2O3; Monoclinic

Phase; Gas-phase Synthesis.

70

Composite Eu2O3/SiO2

particles

Eu2O3/SiO2 Composite

Nanoparticles; Higher-density Core with Lower-density Shell;

Gas-phase Synthesis.Photoluminescence Spectrum: Dominant Peak at ~ 615 nm;

Lifetime ~ 1 ms

71

Lifetimes of 1 ms or more

Biological background typically less than 10 ns

Europium oxide lifetime

Eu:Y2O

3

72

Microwave chemistry and gas phase chemistry used to add protective SiO layer and NH2 functional group

Functionalized nanoparticles

73

Optical properties of Eu2O3 unchanged by surface treatment

Additional phosphors obtained by doping lanthanides into hosts

Used in immunoassay for atrazine

Optical properties of functionalized particles

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

10000000

550 600 650 700 750

Wavelength / nmIn

ten

sity

0

10000

20000

30000

40000

350 400 450 500

Wavelength / nm

Capped with silane

74

80 m channel electrophoresis system

separated two size classes of nanoparticles

can be used for assays

Nanophosphors separated with microchip electrophoresis

75

Europium oxide label in a competitive immunoassay

Competition

Ab

Labeled analyte

76

Europium oxide label in a competitive immunoassay

Separation

Magnetic beadand 2 Ab

77

Europium oxide in a pyrethroid metabolite assay

Competitive assay for 3-phenoxybenzoic acid (PBA) with magnetic separation

Europium oxide particle conjugated to the hapten

IC50

=

2 x 10 –4 ng mL-1

78

New nanoparticle assay formats

Multi wavelength labels can be used to provide an internal standard for sandwich immunoassays

79

Time gated detection

80

Eu phosphors in micro channels

81

Eu lifetime can be greater than 1ms

Fluorescein lifetime of the order of 10 ns

Fluorescence lifetimes

82

MEMS sensors

Quartz transmits UV excitation

Glass blocks excitation light, transmits visible signal

83

Nanoscale materials, engineered from the “bottom up”, can be functionalized for use in biology

Optical properties promise more sensitive detection in complex matrix, possibly reagentless

Eu labels demonstrated in immunoassays with up to 10 4 improvement in detection limits

Other properties of nanoscale materials can be useful (magnetic, thermophoretic, electrophoretic) for separation

Brilliant dust?

Summary

84

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